The present invention relates to compositions for delivering active agents, and particularly biologically or chemically active agents. The compositions comprise a polymeric delivery agent or delivery agent compound which facilitates the delivery of the active agent to a target. These polymeric delivery agents and delivery agent compounds are well suited to form non-covalent mixtures with active agents for administration to animals. Methods for the preparation and for the administration of such compositions are also disclosed.
Conventional means for delivering active agents are often severely limited by biological, chemical, and physical barriers. Typically, these barriers are imposed by the environment through which delivery occurs, the environment of the target for delivery, or the target itself. Biologically or chemically active agents are particularly vulnerable to such barriers.
In the delivery to animals of pharmacological and therapeutic agents, barriers are imposed by the body. Physical barriers such as the skin and various organ membranes are relatively impermeable to certain active agents but must be traversed before reaching a target, such as the circulatory system. Chemical barriers include, but are not limited to, pH variations in the gastrointestinal (GI) tract and degrading enzymes.
Oral delivery of many biologically or chemically active agents would be the route of choice for administration to animals if not for biological, chemical, and physical barriers. Among the numerous agents which are not typically amenable to oral administration are biologically or chemically active peptides, such as calcitonin and insulin; polysaccharides, and in particular mucopolysaccharides including, but not limited to, heparin; heparinoids; antibiotics; and other organic substances. These agents may be rendered ineffective or may be destroyed in the GI tract by acid hydrolysis, enzymes, or the like, or may simply not be absorbed.
Many delivery agents are fairly hydrophobic, whereas many active agents are hydrophilic. The differential aqueous solubility between the delivery agent and the active agent can be problematic in designing commercially acceptable dosage formulations which exhibit biological activity in vivo. Thus, the ability to alter the solubility of a delivery agent would allow one to tailor the delivery agent to meet the needs of the cargo in order to optimize its bioavailability.
The pH within the gastrointestinal tract typically ranges from about 1 to about 8, while many delivery agents remain soluble over a range of only 2-2.5 pH units. During oral delivery, a significant amount of such a delivery agent could precipitate out in the stomach due to the local acidity. The precipitated delivery agent would then be unavailable for delivery of active agent to a point further along the GI tract. Increasing the span of pH solubility of the delivery agent would allow more effective delivery at lower concentrations of delivery agent.
Delivery agents generally tend to self-aggregate into micellular-like structures. The competition between self association and association with the active agent typically results in at least a portion of the delivery agent being unavailable for effective delivery of the active agent. Thus, a corresponding portion of the active agent that was administered may not be effectively delivered to the target. Inhibiting self aggregation of the delivery agent would increase the availability of delivery agent for delivery of the active agent.
Various delivery agents for oral administration of active agents have been developed in recent years. These delivery agents include proteinoids, modified vegetable proteins, acylated or sulfonated amino acids, acylated or sulfonated amino acid ketones, and acylated or sulfonated amino acid aldehydes. See, U.S. Pat. Nos. 5,401,516; 5,443,841; 5,451,410; 5,541,155; 5,629,020; 5,643,957; 5,693,338; 5,709,861; 5,714,167; 5,766,633; 5,773,647; 5,792,451; 5,820,881; 5,863,944; 5,866,536; and RE35,862. These delivery agents promote systemic absorption of active agents in the gastrointestinal tract. The interaction between the delivery agent and the active agent, as well as the interaction between the delivery agent and the cell membrane, may be important for absorption. See, U.S. Pat. No. 5,714,167.
There is a need for delivery agents whose solubility can be modified for a particular need, thereby changing the concentration of soluble delivery agent which is available for delivery of an active agent.
Therefore, there is a need for alternate and improved delivery agents.
The present invention provides polymeric delivery agents which are useful in the delivery of active agents. The polymeric delivery agent comprises a polymer conjugated to a modified amino acid or derivative thereof via a linkage group selected from the group consisting of xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OOCxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94NHCH2xe2x80x94, xe2x80x94CH2NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHCH2xe2x80x94, xe2x80x94CH2NHCOCH2Oxe2x80x94, xe2x80x94OCH2C(O)NHCH2xe2x80x94, xe2x80x94NHC(O)CH2Oxe2x80x94, xe2x80x94OCH2C(O)NHxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, and carbon-carbon bond, with the proviso that the polymeric delivery agent is not a polypeptide or polyamino acid The modified amino acids may be acylated or sulfonated amino acids, ketones or aldehydes of acylated or sulfonated amino acids, salts thereof, or polyamino acids or polypeptides of any of the foregoing.
The polymer may be any polymer including, but not limited to, alternating copolymers, block copolymers and random copolymers, which are safe for use in mammals. Preferred polymers include, but are not limited to, polyethylene; polyacrylates; polymethacrylates; poly(oxyethylene); poly(propylene); polypropylene glycol; polyethylene glycol (PEG) and derivatives thereof, such as PEG-maleic anhydride copolymers; and derivatives and combinations thereof. The molecular weight of the polymer typically ranges from about 100 to abut 200,000 daltons. The molecular weight of the polymer preferably ranges from about 200 to about 10,000 daltons. In one embodiment, the molecular weight of the polymer ranges from about 200 to about 600 daltons and more preferably ranges from about 300 to about 550 daltons.
According to one embodiment, the polymeric delivery agent comprises units having the formula 
or salts thereof where R1 is a modified amino acid which is bonded to the polymer via a linkage group selected from the group consisting of xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OOCxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94CC(O)NHxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94NHCH2xe2x80x94, xe2x80x94CH2NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHCH2xe2x80x94, xe2x80x94CH2NHCOCH2Oxe2x80x94, xe2x80x94OCH2(O)NHCH2xe2x80x94, xe2x80x94NHC(O)CH2Oxe2x80x94, xe2x80x94OCH2C(O)NHxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, and carbon-carbon bond; R2 is H or xe2x80x94CH3; and R19 is H or xe2x80x94COCH. Preferably, R1 is xe2x80x94R3xe2x80x94R4 where R3 is xe2x80x94NHC(O)NHxe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OOCxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94NHCH2xe2x80x94, xe2x80x94CH2NHC(O)Oxe2x80x94, xe2x80x94OC(O)NHCH2xe2x80x94, xe2x80x94CH2NHCOCH2Oxe2x80x94, xe2x80x94OCH2C(O)NHCH2xe2x80x94, xe2x80x94NHC(O)CH2Oxe2x80x94, xe2x80x94OCH2C(O)NHxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, or carbon-carbon bond; and R4 has the formula 
where
R5, R6, R7, R8, and R9 are independently a bond to R3, or H, Cl, Br, F, xe2x80x94OH, xe2x80x94CH3, xe2x80x94CH3, or xe2x80x94(CH2)mCH3;
R10 is a bond to R3 or xe2x80x94COOH, or xe2x80x94C(O)NHxe2x80x94R11xe2x80x94R12 ;
R11 is a substituted or unsubstituted, linear or branched alkylene Slaving a chain length of from about 1 to about 11 or xe2x80x94R13xe2x80x94R14xe2x80x94;
R12 is a bond to R3 or is xe2x80x94COOH, xe2x80x94NH2, xe2x80x94OH, xe2x80x94C(O)xe2x80x94R15, xe2x80x94COOxe2x80x94R15, xe2x80x94NHR15, xe2x80x94OR15, Cl, or Br;
R13 is a substituted or unsubstituted phenylene;
R14 is a substituted or unsubstituted, linear or branched alkylene having a chain length of from about 1 to about 5;
R15 is a bond to R3; and
m is from about 1 to about 4.
Preferably, R4 is selected from the group consisting of 
and salts thereof.
Preferably, R9 is xe2x80x94OCH3 or xe2x80x94OH. According to a preferred embodiment, R10 is xe2x80x94NHxe2x80x94R11xe2x80x94R12 and R11 is xe2x80x94(CH2)7xe2x80x94, xe2x80x94(CH2)9xe2x80x94, xe2x80x94(C6H5)xe2x80x94, xe2x80x94(CH2)xe2x80x94, xe2x80x94(C6H5)xe2x80x94CH2xe2x80x94, or xe2x80x94(CH2)8xe2x80x94NHxe2x80x94C(O)xe2x80x94CH2xe2x80x94.
Another embodiment is a polymeric delivery agent having units of the formula
R16xe2x80x94R24xe2x80x94CH2CH2xe2x80x94R17
or salts thereof where R16 is defined as R1 above; R17 is xe2x80x94OH, xe2x80x94OCH3, or xe2x80x94R18; R18 is defined as R1 above; and R24 is a polymer having units of xe2x80x94(CH2CH2O)xe2x80x94, xe2x80x94(CH(CH3)CH2O)xe2x80x94, or a combination thereof. R24 typically contains from about 3 to about 200 polymeric units. R24 may be a random copolymer or a block copolymer. R18 may be the same or different than R16.
A preferred embodiment of the polymeric delivery agent has units of the formula 
or salts thereof where R16, R17, and R18 are defined as above; R23 is H or xe2x80x94CH3; and n is from about 3 to about 200. Preferably, R16 and R18 are xe2x80x94OOCxe2x80x94R4. According to a preferred embodiment, R17 is xe2x80x94OCH3. According to another preferred embodiment, R16 is xe2x80x94NHC(O)-R4 or xe2x80x94NHC(O)Oxe2x80x94R4 and n ranges from about 4 to about 15.
Yet another embodiment is a polymeric delivery agent having units of the formula 
or salts thereof where R20, R21, and R22 independently are H or are defined as R1 above; a, b, and c independently are integers from about 1 to about 50; and d ranges from about 2 to about 10. Preferably, R20, R21, and R22 independently are xe2x80x94COOxe2x80x94R4. Preferably, d is about 6.
Examples of polymeric delivery agents and units for the polymeric delivery agents include, but are not limited to, 
wherein x is 0.02 to 0.5, preferably 0.05 (Conjugate 1 is a random polymer); 
wherein x is 0.02 to 0.5, preferably 0.06 (Conjugate 1 is a random polymer); 
wherein
k=1-11, preferably 7 or 9
n=10 to 50, preferably 33, and
m=5 to 15, preferably 9.
Conjugate 3 is the rove structure where k=7, n=33 and m=8;
I-COOxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OH Conjugate 4.
I-COOxe2x80x94CH2CH2O(CH2CH2O)3CH2CH2OH Conjugate 5
I-COOxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 6
I-COOxe2x80x94CH2CH2O(CH2CH2O)7CH2CH2OH Conjugate 7
II-COOxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OH Conjugate 8
III-COOxe2x80x94CH2CH2O(CH2CH2O)7CH2CH2OH Conjugate 9
I-COOxe2x80x94CH2CH2O(CH2CH2O)11CH2CH2OH Conjugate 10
I-COOxe2x80x94CH2CH2O(CH2CH2O)102CH2CH2OOC-I Conjugate 11
PEG branched (8 arms): 
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 4 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 12
I-COOxe2x80x94CH2CH2O(CH2CH2O)14CH2CH2OCH3 Conjugate 13
IV-COOxe2x80x94CH2CH2O(CH2CH2O)4CH2CH2OH Conjugate 14
V-COOxe2x80x94CH2CH2O(CH2CH2O)4CH2CH2OH Conjugate 15
IV-COOxe2x80x94CH2CH2O(CH2CH2O)6CH2CH2OH Conjugate 16
I-CH2NHxe2x80x94COxe2x80x94Oxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 17
I-CH2NHxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 18
II-COOxe2x80x94CH2CH2O(CH2CH2O)10CH2CH2OCH3 Conjugate 19
II-COOxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 20
I-COOxe2x80x94CH2CH2O(CH2CH2O)10CH2CH2OCH3 Conjugate 21
I-CH2NHxe2x80x94COxe2x80x94CH2Oxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 22
I-CH2NHxe2x80x94COxe2x80x94CH2Oxe2x80x94CH2CH2O(CH2CH2O)10CH2CH2OCH3 Conjugate 23
VI-COOxe2x80x94CH2CH2O(CH2CH2O)5CH2CH2OCH3 Conjugate 24
VI-COOxe2x80x94CH2CH2O(CH2CH2O)10CH2CH2OCH3 Conjugate 25
VII-COOxe2x80x94CH2CH2O(CH2CH2O)10CH2CH2OCH3 Conjugate 26
VIII-COOxe2x80x94CH2CH2O(CH2CH2O)6CH2CH2OH Conjugate 27
III-COOxe2x80x94CH2CH2O(CH2CH2O)4CH2CH2OH Conjugate 28
III-COOxe2x80x94CH2CH2O(CH2CH2O)6CH2CH2OH Conjugate 29 
PEG branched (8 arms): 
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 6 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 32
I-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)11xe2x80x94CH2CH2OOC-I Conjugate 33
(I-COOH)-5xe2x80x94CH2NHxe2x80x94COxe2x80x94OCH2CH2xe2x80x94(OCH2CH2)43xe2x80x94OCH2CH2xe2x80x94OCH3 Conjugate 34
PEG branched (8 arms): 
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 4 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 35
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 5 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 36
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 7 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 37
The modified amino acid I-COO is attached at the xe2x80x94OH group through an ester linkage at 8 of the 8 xe2x80x9carmsxe2x80x9d. Conjugate 38
I-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)45xe2x80x94CH2CH2OOC-I Conjugate 39
I-NHxe2x80x94COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)42xe2x80x94CH2CH2OCH3 Conjugate 40
I-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)4xe2x80x94CH2CH2OOC-I Conjugate 41
XI-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)4xe2x80x94CH2CH2OH Conjugate 42
X-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)10xe2x80x94CH2CH2OCH3 Conjugate 43
X-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)11xe2x80x94CH2CH2OH Conjugate 44
X-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)20xe2x80x94CH2CH2Oxe2x80x94CO-X conjugate 45
X-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)20xe2x80x94CH2CH2OH Conjugate 46
X-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)11xe2x80x94CH2CH2Oxe2x80x94CO-X Conjugate 47
IX-COOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)5xe2x80x94CH2CH2OCH3 Conjugate 48
The number of polymeric units specified in the aforementioned polymeric delivery are an average number of units. The number of units in the polymers typically may vary by up to about 10%.
Another embodiment provides a composition comprising (A) at least one active agent; and (B) at least one of the aforementioned polymeric delivery agents. The active agent preferably is a biologically or chemically active agent. Methods for the preparation and administration of the composition are also provided. These compositions are useful in the delivery of active agents to selected biological systems and for increasing or improving the bioavailability of the active agent compared to administration of the active agent without the delivery agent.
The invention also includes a method of preparing a polymeric delivery agent by conjugating a modified amino acid to a polymer, via one of the aforementioned linkage groups.
The invention further includes delivery agent compounds having the formulae 
and salts thereof, including but not limited to sodium salts. These delivery agent compounds are useful for facilitating the delivery of an active agent. Another embodiment is a composition comprising one of the aforementioned delivery agent compounds and an active agent.
These compositions may be used to deliver various active agents through or across various biological, chemical, and physical barriers and are particularly suited for delivering active agents that are subject to environmental degradation. The compositions of the subject invention are particularly useful for delivering or administering biologically or chemically active agents to any animals, including but not limited to birds such as chickens; mammals, such as rodents, cows, pigs, dogs, cats, primates, and particularly humans; and insects.
Other advantages of the present invention include the use of easy-to-prepare, inexpensive raw materials. The compositions and the methods of the present invention are cost effective, easy to perform, and amenable to industrial scale up for commercial production.
The presently disclosed compositions deliver active agents, particularly in oral, intranasal, sublingual, intraduodenal, subcutaneous, buccal, intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal, intradermal, parenteral, intravenous, intramuscular and ocular systems as well as traversing the blood-brain barrier. Coadministration of an active agent and a polymer-delivery agent conjugate results in an increased bioavailability of the active agent compared to administration of the active agent alone.
The term xe2x80x9csaltsxe2x80x9d as used in this application includes but are not limited to organic and inorganic salts, for example alkali-metal salts, such as sodium, potassium and lithium; alkaline-earth metal salts, such as magnesium, calcium or barium; ammonium salts; basic amino acids such as lysine or arginine; and organic amines, such as dimethylamine or pyridine. Preferably, the salts are sodium salts.
Active agents suitable for use in the present invention include biologically active agents, and chemically active agents, including, but not limited to, pesticides, pharmacological agents, and therapeutic agents.
For example, biologically active agents suitable for use in the present invention include, but are not limited to, proteins, polypeptides; peptides; hormones; polysaccharides, and particularly muco-polysaccharides and mixtures thereof; carbohydrates; lipids; other organic compounds; and particularly compounds which by themselves do not pass (or which pass only a fraction of the administered dose) through the gastrointestinal mucosa and/or are susceptible to chemical and/or enzymatic cleavage by acids and enzymes in the gastro-intestinal tract; or any combination thereof.
Further examples include, but are not limited to, the following, including synthetic, natural or recombinant sources thereof: growth hormones, including human growth hormones (hGH), recombinant human growth hormone (rhGH), bovine growth hormones, and porcine growth hormones; growth hormone-releasing hormones; interferons, including xcex1, xcex2 and xcex3; interleukin-1; interleukin-2; insulin, including porcine, bovine, human and human recombinant, optionally having counter ions including sodium, zinc, calcium and ammonium; insulin-like growth factor, including IGF-1; heparin, including unfractionated heparin, heparinoids, dermatans, chondroitins, low molecular weight heparin, very low molecular weight heparin and ultra low molecular weight haparin; calcitonin, including salmon, eel and human; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomycin; desferroxamine (DFO); parathyroid hormone (PTH), including its fragments; antimicrobials, including anti-fungal agents; vitamins; analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these compounds; or any combination thereof.
The modified amino acid may be an N-acylated or sulfonated amino acid, a ketone or aldehyde of an acylated or sulfonated amino acid, salts thereof, and a polyamino acid or polypeptide which includes any of the foregoing.
N-acylated and sulfonated amino acids, poly amino acids, and peptides include, but are not limited to, amino acids which have been N-acylated or sulfonated, and poly amino acids and peptides in which at least one amino acid has been modified, by acylating or sulfonating at least one free amine group with an acylating or sulfonating agent which reacts with at least one of the free amine groups present.
Preferably, the modified amino acids comprise one of the following structures: 
and salts thereof, including but not limited to sodium salts.
The modified amino acids may be in the form of salts. Salts include but are not limited to organic and inorganic salts, for example alkali-metal salts, such as sodium, potassium and lithium; alkaline-earth metal salts, such as magnesium, calcium or barium; ammonium salts; basic amino acids such as lysine or arginine; and organic amines, such as dimethylamine or pyridine. Preferably, the salts are sodium salts.
An amino acid is any carboxylic acid having at least one free amine group and includes naturally occurring and synthetic amino acids. Poly amino acids are either peptides (which are two or more amino acids joined by a peptide bond) or are two or more amino acids linked by a bond formed by other groups which can be linked, e.g. an ester, anhydride, or an anhydride linkage. Peptides can vary in length from dipeptides with two amino acids to poly peptides with several hundred amino acids. One or more of the amino acid or peptide units may be acylated or sulfonated.
N-acylated or sulfonated amino acids are typically prepared by modifying the amino acid or an ester thereof Many of these compounds are prepared by acylation or sulfonation with agents having the formula
xe2x80x83Xxe2x88x92Yxe2x88x92R
wherein:
R is the appropriate radical to yield the modification indicated in the final product,
Y is Cxe2x95x90O or SO2, and
X is a leaving group.
Typical leaving groups include, but are not limited to, halogens such as, for example, chlorine, bromine, and iodine. Additionally, the corresponding anhydrides are modifying agents.
N-acylated or sulfonated amino acids can be readily prepared from amino acids by methods within the skill of those in the art based upon the present disclosure. For example, N-acylated or sulfonated amino acids may be derived from aminobutyric acid, aminocaproic acid, and aminocaprylic acid. Further, the N-acylated or sulfonated amino acid above may be prepared by reacting the single amino acid with the appropriate modifying agent which reacts with a free amino moiety present in the amino acids to form amides. Protecting groups may be used to avoid unwanted side reactions as would be known to those skilled in the art.
The amino acid can be dissolved in aqueous alkaline solution of a metal hydroxide, e.g., sodium or potassium hydroxide, and heated at a temperature ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably between about 10xc2x0 C. and about 40xc2x0 C., for a period ranging between about 2 hour and about 4 hours, preferably about 2.5 hours. The amount of alkali employed per equivalent of NH2 groups in the amino acid generally ranges between about 1.25 and about 3 mmole, preferably between about 1.5 and about 2.25 mmole per equivalent of NH2. The pH of the solution generally ranges between about 8 and about 13, preferably ranging between about 10 and about 12.
Thereafter, the appropriate amino modifying agent is added to the amino acid solution while stirring. The temperature of the mixture is maintained at a temperature generally ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably between about 10xc2x0 C. and about 40xc2x0 C., for a period ranging between about 1 and about 4 hours. The amount of amino modifying agent employed in relation to the quantity of amino acid is based on the moles of total free NH2 in the amino acid. In general, the amino modifying agent is employed in an amount ranging between about 0.5 and about 2.5 mole equivalents, preferably between about 0.75 and about 1.25 equivalents, per molar equivalent of total NH2 group in the amino acid.
The reaction is quenched by adjusting the pH of the mixture with a suitable acid, e.g., concentrated hydrochloric acid, until the pH reaches between about 2 and about 3. The mixture separates on standing at room temperature to form a transparent upper layer and a white or off-white precipitate. The upper layer is discarded, and the N-acylated or sulfonated amino acid is collected from the lower layer by filtration or decantation. The crude N-acylated or sulfonated amino acid is then dissolved in water at a pH ranging between about 9 and about 13, preferably between about 11 and about 13. Insoluble materials are removed by filtration and the filtrate is dried in vacuo The yield of N-acylated or sulfonated amino acid generally ranges between about 30 and about 60%, and usually about 45%.
If desired, amino acid esters, such as, for example benzyl, methyl, or ethyl esters of amino acid compounds, may be used to prepare the N-acylated or sulfonated amino acids of the invention. The amino acid ester, dissolved in a suitable organic solvent such as dimethylformamide, pyridine, or tetrahydrofuran is reacted with the appropriate amino modifying agent at a temperature ranging between about 5xc2x0 C. and about 70xc2x0 C., preferably about 25xc2x0 C., for a period ranging between about 7 and about 24 hours. The amount of amino modifying agent used relative to the amino acid ester is the same as described above for amino acids. This reaction may be carried out with or without a base such as, for example, triethylamine or diisopropylethylamine.
Thereafter, the reaction solvent is removed under negative pressure and the ester functionality is removed by hydrolyzing the N-acylated or sulfonated amino acid ester with a suitable alkaline solution, e.g. 1N sodium hydroxide, at a temperature ranging between about 50xc2x0 C. and about 80xc2x0 C., preferably about 70xc2x0 C., for a period of time sufficient to hydrolyze off the ester group and form the N-acylated or sulfonated amino acid having a free carboxyl group. The hydrolysis mixture is then cooled to room temperature and acidified, e.g. aqueous 25% hydrochloric acid solution, to a pH ranging between about 2 and about 2.5. The N-acylated or sulfonated amino acid precipitates out of solution and is recovered by conventional means such as filtration or decantation. Benzyl esters may be removed by hydrogenation in an organic solvent using a transition metal catalyst.
The N-acylated or sulfonated amino acid may be purified by recrystallization or by fractionation on solid column supports
Suitable recrystallization solvent systems include acetonitrile, methanol and tetrahydrofuran. Fractionation may be performed on suitable solid column supports such as alumina, using methanol/n-propanol mixtures as the mobile phase; reverse phase column supports using trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and ion exchange chromatography using water as the mobile phase. When anion exchange chromatography is performed, preferably a subsequent 0-500 mm sodium chloride gradient is employed.
The polymers of the present invention may be natural or synthetic and comprise two or more monomers. The monomers may be the same or different, and the polymer may be linear or non-linear Polymers include but are not limited to branched or cyclic polymers. The polymers may be copolymers including two or more different monomers, or homo-polymers including a single-type of monomeric repeat. Further, polymers may be random or alternating, directed, bifunctional, polyfunctional, cross-linked, regular lattice, intermittent lattice, or amorphous.
The polymer may be any polymer including, but not limited to, alternating copolymers, block copolymers and random copolymers, which are safe for use in mammals. Preferred polymers include, but are not limited to, polyethylene; polyacrylates; polymethacrylates; poly(oxyethylene); poly(propylene); polypropylene glycol; polyethylene glycol (PEG) and derivatives thereof, such as PEG-maleic anhydride copolymers; and derivatives and combinations thereof. The molecular weight of the polymer typically ranges from about 100 to about 200,000 daltons The molecular weight of the polymer preferably ranges from about 200 to about 10,000 daltons. In one embodiment, the molecular weight of the polymer ranges from about 200 to about 600 daltons and more preferably ranges from about 300 to about 550 daltons.
Polymers may be in the form of one or more salts. Salts include but are not limited to organic and inorganic salts, for example alkali-metal salts, such as sodium, potassium and lithium; alkaline-earth metal salts, such as magnesium, calcium or barium; ammonium salts; basic amino acids such as lysine or arginine; and organic amines, such as dimethylamine or pyridine. Preferably, the salts are sodium salts.
One or more of the modified amino acids may be conjugated (covalently attached) to one or more of the monomeric units of the polymer via one of the aforementioned linkage groups.
Many of the polymeric delivery agents have solubility greater than about 200 mg/mL, and have greater solubility than the corresponding modified amino acids alone. However, like most poloxamers, the solubility of PEG conjugates decreases at higher temperatures and can be characterized by the cloud point or lower critical solution temperature (LCST). The LCST is dependent on the ratio of hydrophilic/hydiophobic units in the conjugate and can be changed easily.
In general, the polymeric delivery agents of the present invention may be prepared as follows. For vinyl polymeric delivery agents, such as PAA and PAA/MA polymers, the polymer and modified amino acids may each be separately dissolved in an appropriate solvent, e.g., dimethyl formamide (DMF), to yield solutions A and B, respectively. Solution B is warmed to about 60-70xc2x0 C., in the presence of a base, e.g. triethylamine. Solution B is then added to solution A and the mixture is stirred at room temperature for 24 hours. The polymeric delivery agents precipitates with the addition of dilute acid or base and is collected by centrifugation. The polymeric delivery agents is then hydrolyzed, dialyzed against water, and lyophilized.
The resultant polymeric delivery agents may be analyzed by Size Exclusion Chromatography (SEC) in order to determine the approximate molecular weight of the polymer and the nitrogen content of the conjugate may be used to approximate the amount of modified amino acid bound to the polymer in the polymeric delivery agent. Preferably, there is between about 5 and 15% w/w bound modified amino acid in the polymeric delivery agent, and more preferably, there is between about 10 and 15% w/w bound modified amino acid unit in the polymeric delivery agent.
For PEG delivery agents with ester linkages, a carboxyl-containing deliver agent reacts with PEG or PEG methyl ether in toluene at 150-160xc2x0 C. in the presence of p-toluene sulfonic acid as a catalyst for 3-4 hours. Water generated by the reaction is removed with a Dean-Stark trap. Reverse phase HPLC is used to monitor the reaction. The reaction mixture is washed with saturated NaHCO3 water solution to remove unreacted starting materials and the catalyst. The polymeric delivery agents are obtained after evaporation of toluene. The structure is further confirmed by nitrogen analysis and 1H NMR.
PEG delivery agents with amide, amino or urethane linkages may be prepared by reaction of an amino-containing modified amino acids with an appropriately activated polyethylene glycol in pyridine at 70-80xc2x0 C. for 4-5 hours and at room temperature for about 24 hours. Pyridine is then removed by evaporation under reduced pressure. The residue is then dissolved in an organic solvent, e.g., methylene chloride, and the solution is washed with dilute HCl aq., NaCl aq., and NaHCO3 aq. respectively to remove impurities. Reverse phase RPLC is used to monitor both the reaction and the work-up process. The polymeric delivery agents is obtained after evaporation of the organic solvent. The structure is further confirmed by nitrogen analysis and 1H NMR.
In order to prepare PEG delivery agents with urea linkages, a two step process was used. First, a urethane derivative based on the reaction of an amino terminated hydrophobic compound and 4-nitrophenyl chloroformate is prepared. The reaction is very fast and carried out at room temperature in pyridine solution. The intermediate urethane derivative contains a good leaving group that can be eliminated on attack of nucleophilic agents. When this intermediate is reacted with an amino-terminated PEG, both 4-nitrophenol and the PEG adduct with urea linkage were formed.
The compositions of the present invention may include one or more active agents. In one embodiment, the polymeric delivery agents or delivery agent compounds of the present invention (collectively xe2x80x9cdelivery agentsxe2x80x9d) may be used directly as a delivery agent by simply mixing one or more delivery agent with the active agent prior to administration. The administration mixtures may be prepared by mixing an aqueous solution of the delivery agent with an aqueous solution of the active ingredient, just prior to administration.
Alternatively, the delivery agent and the active agent can be admixed during the manufacturing process. The solutions may optionally contain additives such as phosphate buffer salts, citric acid, acetic acid, gelatin, and gum acacia.
Stabilizing additives may be incorporated into the delivery agent solution. With some active agents, the presence of such additives promotes the stability and dispersibility of the agent in solution. The stabilizing additives may be employed at a concentration ranging between about 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable, but non-limiting, examples of stabilizing additives include gum acacia, gelatin, methyl cellulose, polyethylene glycol, carboxylic acids and salts thereof, and polylysine. The preferred stabilizing additives are gum acacia, gelatin and methyl cellulose.
The amount of active agent is an amount effective to accomplish the purpose of the particular active agent. The amount in the composition typically is a pharmacologically or biologically effective amount. However, the amount can be less than a pharmacologically or biologically effective amount when the composition is used in a dosage unit form, such as a solid, such as a capsule, a tablet, or a powder, or a liquid, because the dosage unit form may contain a multiplicity of delivery agent or active agent compositions or may contain a divided pharmacologically or biologically effective amount. The total effective amounts can then be administered in cumulative units containing, in total, pharmacologically or biologically or chemically active amounts of biologically or pharmacologically active agent.
The total amount of active agent to be used can be determined by those skilled in the art. However, because the presently disclosed delivery agents provide efficient delivery, lower amounts of biologically or chemically active agent than chose used in prior dosage unit forms or delivery systems may be administered to the subject, while still achieving the same blood levels and therapeutic effects.
The amount of delivery agents in the present composition is a delivery effective amount and can be determined for any particular deliver agents or active agent by methods known to those skilled in the art. It will be this amount effective for delivery of the active agent by the chosen route of delivery.
Dosage unit forms can also include any of excipients; diluents; disintegrants; lubricants; plasticizers; colorants; and dosing vehicles, including, but not limited to water, 1,2-propane diol, ethanol, olive oil, or any combination thereof.
Administration of the present compositions or dosage unit forms preferably is oral, intracolonic or intraduodenal. Particularly, the compositions of the present invention are useful in orally administering active agents, especially those that are not ordinarily orally deliverable.
The delivery compositions of the present invention may also include one or more enzyme inhibitors. Such enzyme inhibitors include, but are not limited to, compounds such as actinonin or epiactinonin and derivatives thereof. Other enzyme inhibitors include, but are not limited to, aprotinin (Trasylol) and Bowman-Birk inhibitor.
The compositions of the subject invention are useful for administering biologically or chemically active agents to animals. The system is particularly advantageous for delivering biologically or chemically active agents which would otherwise be destroyed or rendered less effective by conditions encountered before the active agent has reached its target zone (i.e. the area in which the active agent of the delivery composition are to be released) and within the body of the animal to which they are administered.